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We here describe a multimodality neuroimaging containing data from healthy volunteers and patients, acquired within the Lundbeck Foundation Center for Integrated Molecular Brain Imaging (Cimbi) in Copenhagen, Denmark. The data is of particular relevance for neurobiological research questions related to the serotonergic transmitter system with its normative data on the serotonergic subtype receptors 5-HT1A, 5-HT1B, 5-HT2A, and 5-HT4 and the 5-HT transporter (5-HTT), but can easily serve other purposes.The Cimbi database and Cimbi biobank were formally established in 2008 with the purpose to store the wealth of Cimbi-acquired data in a highly structured and standardized manner in accordance with the regulations issued by the Danish Data Protection Agency as well as to provide a quality-controlled resource for future hypothesis-generating and hypothesis-driven studies.The Cimbi database currently comprises a total of 1100 PET and 1000 structural and functional MRI scans and it holds a multitude of additional data, such as genetic and biochemical data, and scores from 17 self-reported questionnaires and from 11 neuropsychological paper/computer tests. The database associated Cimbi biobank currently contains blood and in some instances saliva samples from about 500 healthy volunteers and 300 patients with e.g., major depression, dementia, substance abuse, obesity, and impulsive aggression. Data continue to be added to the Cimbi database and biobank.

Pretargeted radioimmunoimaging has been shown to enhance tumor-to-background ratios by up to 125-fold at early time points, leading to more efficient and less toxic radionuclide therapies, particularly with shorter half-lives such as astatine-211 ( 211 At). The tetrazine ligation is the most utilized bioorthogonal reaction in these strategies, making tetrazines ideal for 211 At labeling and controlling the biodistribution. We developed a 211 At-labeled pretargeting agent for alpha-radionuclide therapy, achieving a radiochemical yield of approximately 65% and purity over 99%. Our results showed higher tumor-to-blood ratios within the first 24 h compared to directly labeled monoclonal antibodies. This suggests that pretargeted therapy may deliver better tumor doses than conventional methods, although the deastatination observed will need to be addressed in future tetrazine developments.

The serotonin 7 receptor (5-HT 7 R) regulates various processes in the central nervous system, including mood, learning, and circadian rhythm control, among others. Receptor activation can lead to activation of the Gα s protein and a subsequent increase of intracellular cyclic adenosine monophosphate (cAMP). Receptor interaction with inverse agonists results in a decrease of basal cAMP levels and therefore a downstream effect of reduced neuronal excitability and neurotransmission. Recently, pellotine ( 1a ), a Lophophora alkaloid, was unexpectedly shown to be an inverse agonist of the 5-HT 7 R. Therefore, we evaluated close analogs of compound 1a , both naturally occurring and synthetic analogs, as inverse agonists of the 5-HT 7 R. Functional evaluation in a GloSensor cAMP assay revealed a preference for an 8-hydroxy-6,7-dimethoxy substitution pattern over 6,7,8-trimethoxy analogs or 8-hydroxy-6,7-methylenedioxy analogs. This was supported by molecular dynamics simulations, where the 8-hydroxy substitution allowed more robust interaction with the 5-HT 7 R, which correlated with inverse agonism efficacy. Additionally, N -methylation (as in 1a ) improved the potency of the evaluated analogs. In this series, the most potent inverse agonist was anhalidine ( 1b ) (EC 50 = 219 nM, E max = -95.4%), which lacks the 1-methyl, compared to pellotine ( 1a ), and showed a 2-fold higher functional potency. Altogether, these results provide key insights for the further development of potent low molecular weight inverse agonists of the 5-HT 7 R.

The absence of universal and expedient labeling procedures hampers the widespread application of peptides as molecular tracers for positron emission tomography (PET). In this work, we have developed a proof-of-concept direct radiofluorination procedure for peptides using conventional solid-phase peptide synthesis. The method is compatible with all standard amino acids and enables the generation of a range of peptide-based radiopharmaceuticals archieving radiochemical yields of up to 30 % and radiochemical purities above 95 %., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Organometallics can be used as precursors for electrophilic 211 At-statinations. In this report, we compared the potential of aryl trimethylsilanes, -germanes, or -stannanes to be used as precursors to 211 At-label highly reactive tetrazines. Tetrazines can be used for pretargeted radioligand therapies or be applied as synthons to radiolabel rapidly and orthogonally a broad set of precursors such as peptides, mAbs or nanomedicines. All precursors could successfully be synthesized and radiolabeled. The reactivity of organogermanium reagents ranged between those of respective organotin and organosilicon precursors. Moreover, organogermanium reagents proved promising for accessing more complex and polar tetrazine scaffolds. In contrast to organotin derivatives, the use of protecting groups could be avoided for organogermanium and -silicon precursors. The developed 211 At-labeled tetrazines could be labeled in radiochemical conversions of 60-90 %. Organogermanium and -silicon precursors were clearly advantaged as additional deprotection steps could avoided. Reported labeling procedures allow astatinations of highly reactive tetrazines to be used for pretargeted approaches or to applied as highly reactive synthons to label the next-generation of 211 At-labeled radiopharmaceuticals., (© 2024 The Author(s). Chemistry - A European Journal published by Wiley-VCH GmbH.)

Polar BODIPY-tetrazine dyes were developed and clicked in vivo to a preaccumulated trans -cyclooctene-modified anti-TAG72 monoclonal antibody CC49 (CC49-TCO). The in vivo click performance was evaluated using an in-house developed ex vivo blocking assay. All tested polar BODIPY structures exhibited excellent in vivo binding, confirming that the turn-on tetrazine dyes successfully clicked in vivo to pretargeted CC49-TCO. Fluorescence imaging showed high tumor-to-muscle ratios of 4:1. This proof-of-concept study indicates that the pretargeting concept based on turn-on probes could be used for cancer treatments, such as photodynamic or -thermal therapy., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

The use of fibroblast activation protein inhibitors (FAPis) for positron emission tomography (PET) imaging in cancer has garnered significant interest in recent years, yielding promising results in preclinical and clinical settings. FAP is predominantly expressed in pathological conditions such as fibrosis and cancer, making it a compelling target. An optimized approach involves using FAPi homodimers as PET tracers, which enhance tumor uptake and retention, making them more effective candidates for therapy. Here, a UAMC-1110 inhibitor-based homodimer, DOTAGA-Glu(FAPi) 2 , was synthesized and radiolabeled with gallium-68, and its efficacy was evaluated in vivo for PET imaging in an endogenously FAP-expressing xenografted mouse model, U87MG. Notably, 45 min post-injection, the mean uptake of [ 68 Ga]Ga-DOTAGA-Glu(FAPi) 2 was 4.7 ± 0.5% ID/g in the tumor with low off-target accumulation. The ex vivo analysis of the FAP expression in the tumors confirmed the in vivo results. These findings highlight and confirm the tracer's potential for diagnostic imaging of cancer and as a theranostic companion.

A set of 211 At-astatoarenes were synthesized from corresponding trimethylgermyl arenes with an average radiochemical conversion (RCC) of ca. 50 % for electron-rich and approx. 70 % in case of electron-deficient arenes. Both electron rich and electron poor substrates were successfully radiolabeled at room temperature (RT) using relatively low precursor amounts (0.15 μmol/0.02 mL solvent (7.5 mM)). Ready access to ortho-, para- and meta- astatinated arenes was achievable. Optimized reaction conditions were successfully applied to label a poly (ADP-ribose) polymerase (PARP) inhibitor with a RCC of approx. 50 %. We believe that trimethylgermyl derivatives are a viable addition to the astatination precursor toolbox and facilitate astatination of arenes. The developed labeling method should easily be applicable for productions under good manufacturing practice (GMP)., (© 2024 The Authors. ChemPlusChem published by Wiley-VCH GmbH.)

The postsynaptic density (PSD) comprises numerous scaffolding proteins, receptors, and signaling molecules that coordinate synaptic transmission in the brain. Postsynaptic density protein 95 (PSD-95) is a master scaffold protein within the PSD and one of its most abundant proteins and therefore constitutes a very attractive biomarker of PSD function and its pathological changes. Here, we exploit a high-affinity inhibitor of PSD-95, AVLX-144, as a template for developing probes for molecular imaging of the PSD. AVLX-144-based probes were labeled with the radioisotopes fluorine-18 and tritium, as well as a fluorescent tag. Tracer binding showed saturable, displaceable, and uneven distribution in rat brain slices, proving effective in quantitative autoradiography and cell imaging studies. Notably, we observed diminished tracer binding in human post-mortem Parkinson's disease (PD) brain slices, suggesting postsynaptic impairment in PD. We thus offer a suite of translational probes for visualizing and understanding PSD-related pathologies.

Background: The development of bispecific antibodies that can traverse the blood-brain barrier has paved the way for brain-directed immunotherapy and when radiolabelled, immunoPET imaging. The objective of this study was to investigate how indium-111 ( 111 In) radiolabelling with compatible chelators affects the brain delivery and peripheral biodistribution of the bispecific antibody RmAb158-scFv8D3, which binds to amyloid-beta (Aβ) and the transferrin receptor (TfR), in Aβ pathology-expressing tg-ArcSwe mice and aged-matched wild-type control mice., Methods: Bispecific RmAb158-scFv8D3 (biAb) was radiolabelled with 111 In using CHX-A"-DTPA, DOTA, or DOTA-tetrazine (DOTA-Tz). Affinity toward TfR and Aβ, as well as stability, was investigated in vitro . Mice were then intravenously administered with the three different radiolabelled biAb variants, and blood samples were collected for monitoring pharmacokinetics. Brain concentration was quantified after 2 and 72 h, and organ-specific retention was measured at 72 h by gamma counting. A subset of mice also underwent whole-body Single-photon emission computed tomography (SPECT) scanning at 72 h after injection. Following post-mortem isolation, the brains of tg-ArcSwe and WT mice were sectioned, and the spatial distribution of biAb was further investigated with autoradiography., Results: All three [ 111 In]biAb variants displayed similar blood pharmacokinetics and brain uptake at 2 h after administration. Radiolabelling did not compromise affinity, and all variants showed good stability, especially the DOTA-Tz variant. Whole-body SPECT scanning indicated high liver, spleen, and bone accumulation of all [ 111 In]biAb variants. Subsequent ex vivo measurement of organ retention confirmed SPECT data, with retention in the spleen, liver, and bone - with very high bone marrow retention. Ex vivo gamma measurement of brain tissue, isolated at 72 h post-injection, and ex vivo autoradiography showed that WT mice, despite the absence of Aβ, exhibited comparable brain concentrations of [ 111 In]biAb as those found in the tg-ArcSwe brain., Conclusions: The successful 111 In-labelling of biAb with retained binding to TfR and Aβ, and retained ability to enter the brain, demonstrated that 111 In can be used to generate radioligands for brain imaging. A high degree of [ 111 In]biAb in bone marrow and intracellular accumulation in brain tissue indicated some off-target interactions or potential interaction with intrabrain TfR resulting in a relatively high non-specific background signal., Competing Interests: The authors report no conflict of interest., (© 2024 The Author(s).)

DOTATATE is a somatostatin peptide analog used in the clinic to detect somatostatin receptors which are highly expressed on neuroendocrine tumors. Somatostatin receptors are found naturally in the intestines, pancreas, lungs, and brain (mainly cortex). In vivo measurement of the somatostatin receptors in the cortex has been challenging because available tracers cannot cross the blood-brain barrier (BBB) due to their intrinsic polarity. A peptide called melittin, a main component of honeybee venom, has been shown to disrupt plasma membranes and increase the permeability of biological membranes. In this study, we assessed the feasibility of using melittin to facilitate the passage of [ 64 Cu]Cu-DOTATATE through the BBB and its binding to somatostatin receptors in the cortex. Evaluation included in vitro autoradiography on Long Evans rat brains to estimate the binding affinity of [ 64 Cu]Cu-DOTATATE to the somatostatin receptors in the cortex and an in vivo evaluation of [ 64 Cu]Cu-DOTATATE binding in NMRI mice after injection of melittin. This study found an in vitro B max  = 89 ± 4 nM and K D  = 4.5 ± 0.6 nM in the cortex, resulting in a theoretical binding potential (BP) calculated as B max /K D  ≈ 20, which is believed suitable for in vivo brain PET imaging. However, the in vivo results showed no significant difference between the control and melittin injected mice, indicating that the honeybee venom failed to open the BBB. Additional experiments, potentially involving faster injection rates are required to verify that melittin can increase brain uptake of non-BBB permeable PET tracers. Furthermore, an evaluation of whether a venom with a narrow therapeutic range can be used for clinical purposes needs to be considered., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Pretargeting, which is the separation of target accumulation and the administration of a secondary imaging agent into two sequential steps, offers the potential to improve image contrast and reduce radiation burden for nuclear imaging. In recent years, the tetrazine ligation has emerged as a promising approach to facilitate covalent pretargeted imaging due to its unprecedented kinetics and bioorthogonality. Pretargeted bone imaging with TCO-modified alendronic acid (Aln-TCO) is an attractive model that allows the evaluation of tetrazines in healthy animals without the need for complex disease models or targeting regimens. Recent structure-activity relationship studies of tetrazines evaluated important parameters for the design of potent tetrazine-radiotracers for pretargeted imaging. However, limited information is available for 99mTc-labeled tetrazines. In this study, four tetrazines intended for labeling with fac-[99mTc(OH2)3 (CO)3]+ were synthesized and evaluated using an Aln-TCO mouse model. 3,6-bis(2-pyridyl)-1,2,4,5-Tz without additional linker showed higher pretargeted bone uptake and less background activity compared to the same scaffold with a PEG8 linker or 3-phenyl-1,2,4,5-Tz-based compounds. Additionally, improved bone/blood ratios were observed in pretargeted animals compared to animals receiving directly labeled Aln-TCO. The results of this study implicate 3,6-bis(2-pyridyl)-1,2,4,5-Tz as a promising scaffold for potential 99mTc-labeled tetrazines., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Bohrmann et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The application of prostate-specific membrane antigen (PSMA)-targeted α-therapy is a promising alternative to β - -particle-based treatments. 211 At is among the potential α-emitters that are favorable for this concept. Herein, 211 At-based PSMA radiopharmaceuticals were designed, developed, and evaluated. Methods: To identify a 211 At-labeled lead, a surrogate strategy was applied. Because astatine does not exist as a stable nuclide, it is commonly replaced with iodine to mimic the pharmacokinetic behavior of the corresponding 211 At-labeled compounds. To facilitate the process of structural design, iodine-based candidates were radiolabeled with the PET radionuclide 68 Ga to study their preliminary in vitro and in vivo properties before the desired 211 At-labeled lead compound was formed. The most promising candidate from this evaluation was chosen to be 211 At-labeled and tested in biodistribution studies. Results: All 68 Ga-labeled surrogates displayed affinities in the nanomolar range and specific internalization in PSMA-positive LNCaP cells. PET imaging of these compounds identified [ 68 Ga]PSGa- 3 as the lead compound. Subsequently, [ 211 At]PSAt- 3 -Ga was synthesized in a radiochemical yield of 35% and showed tumor uptake of 19 ± 8 percentage injected dose per gram of tissue (%ID/g) at 1 h after injection and 7.6 ± 2.9 %ID/g after 24 h. Uptake in off-target tissues such as the thyroid (2.0 ± 1.1 %ID/g), spleen (3.0 ± 0.6 %ID/g), or stomach (2.0 ± 0.4 %ID/g) was low, indicating low in vivo deastatination of [ 211 At]PSAt- 3 -Ga. Conclusion: The reported findings support the use of iodine-based and 68 Ga-labeled variants as a convenient strategy for developing astatinated compounds and confirm [ 211 At]PSAt- 3 as a promising radiopharmaceutical for targeted α-therapy., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

Background: The brain is a challenging target for antibody-based positron emission tomography (immunoPET) imaging due to the restricted access of antibody-based ligands through the blood-brain barrier (BBB). To overcome this physiological obstacle, we have previously developed bispecific antibody ligands that pass through the BBB via receptor-mediated transcytosis. While these radiolabelled ligands have high affinity and specificity, their long residence time in the blood and brain, typical for large molecules, poses another challenge for PET imaging. A viable solution could be a two-step pre-targeting approach which involves the administration of a tagged antibody that accumulates at the target site in the brain and then clears from the blood, followed by administration of a small radiolabelled molecule with fast kinetics. This radiolabelled molecule can couple to the tagged antibody and thereby make the antibody localisation visible by PET imaging. The in vivo linkage can be achieved by using the inverse electron demand Diels-Alder reaction (IEDDA), with trans-cyclooctene (TCO) and tetrazine groups participating as reactants. In this study, two novel 18 F-labelled tetrazines were synthesized and evaluated for their potential use as pre-targeting imaging agents, i.e., for their ability to rapidly enter the brain and, if unbound, to be efficiently cleared with minimal background retention., Results: The two compounds, a methyl tetrazine [ 18 F]MeTz and an H-tetrazine [ 18 F]HTz were radiolabelled using a two-step procedure via [ 18 F]F-Py-TFP synthesized on solid support followed by amidation with amine-bearing tetrazines, resulting in radiochemical yields of 24% and 22%, respectively, and a radiochemical purity of > 96%. In vivo PET imaging was performed to assess their suitability for in vivo pre-targeting. Time-activity curves from PET-scans showed [ 18 F]MeTz to be the more pharmacokinetically suitable agent, given its fast and homogenous distribution in the brain and rapid clearance. However, in terms of rection kinetics, H-tetrazines are advantageous, exhibiting faster reaction rates in IEDDA reactions with dienophiles like trans-cyclooctenes, making [ 18 F]HTz potentially more beneficial for pre-targeting applications., Conclusion: This study demonstrates a significant potential of [ 18 F]MeTz and [ 18 F]HTz as agents for pre-targeted PET brain imaging due to their efficient brain uptake, swift clearance and appropriate chemical stability., (© 2024. The Author(s).)

The σ-1 receptor is a non-opioid transmembrane protein involved in various human pathologies including neurodegenerative diseases, inflammation, and cancer. The previously published ligand [ 18 F]FTC-146 is among the most promising tools for σ-1 molecular imaging by positron emission tomography (PET), with a potential for application in clinical diagnostics and research. However, the published six- or four-step synthesis of the tosyl ester precursor for its radiosynthesis is complicated and time-consuming. Herein, we present a simple one-step precursor synthesis followed by a one-step fluorine-18 labeling procedure that streamlines the preparation of [ 18 F]FTC-146. Instead of a tosyl-based precursor, we developed a one-step synthesis of the precursor analog AM-16 containing a chloride leaving group for the S N 2 reaction with 18 F-fluoride. 18 F-fluorination of AM-16 led to a moderate decay-corrected radiochemical yield (RCY = 7.5%) with molar activity (A m ) of 45.9 GBq/μmol. Further optimization of this procedure should enable routine radiopharmaceutical production of this promising PET tracer., (© 2024 The Authors. Journal of Labelled Compounds and Radiopharmaceuticals published by John Wiley & Sons Ltd.)

Brain pretargeted nuclear imaging for the diagnosis of various neurodegenerative diseases is a quickly developing field. The tetrazine ligation is currently the most explored approach to achieve this goal due to its remarkable properties. In this work, we evaluated the performance of F-537-Tetrazine, previously developed by Biogen, and N-(3-[ 18 F]fluoro-5-(1,2,4,5-tetrazin-3-yl)benzyl)propan-1-amine, previously developed in our group, thereby allowing for the direct comparison of these two imaging probes. The evaluation included synthesis, radiolabeling and a comparison of the physicochemical properties of the compounds. Furthermore, their performance was evaluated by in vitro and in vivo pretargeting models. This study indicated that N-(3-[ 18 F] fluoro-5-(1,2,4,5-tetrazin-3-yl)benzyl)propan-1-amine might be more suited for brain pretargeted imaging., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Small-molecular fibroblast activation protein inhibitor (FAPI)-based tracer have been shown to be promising Positron Emission Tomography (PET) 68 Ga-labeled radiopharmaceuticals to image a variety of tumors including pancreatic, breast, and colorectal cancers, among others. In this study, we developed a novel 18 F-labeled FAPI derivative. [ 18 F]6 was labeled using a synthon approach based on the tetrazine ligation. It showed subnanomolar affinity for the FAP protein and a good selectivity profile against known off-target proteases. Small animal PET studies revealed high tumor uptake and good target-to-background ratios. [ 18 F]6 was excreted via the liver. Overall, [ 18 F]6 showed promising characteristics to be used as a PET tracer and could serve as a lead for further development of halogen-based theranostic FAPI radiopharmaceuticals., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Matthias M. herth reports a relationship with Tetrakit Technologies that includes: board membership, consulting or advisory, and equity or stocks. Christian B. M. Poulie reports a relationship with Tetrakit Technologies that includes: board membership, employment, and equity or stocks. Vladimir Shalgunov reports a relationship with Tetrakit Technologies that includes: board membership, employment, and equity or stocks. Umberto Maria Battisti reports a relationship with Tetrakit technologies that includes: board membership, employment, and equity or stocks., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

CRANAD-102, a selective near-infrared fluorescent tracer targeting soluble amyloid-β (Aβ) species, has recently attracted attention due to its potential to be used as a diagnostic tool for early stages of Alzheimer's disease (AD). Development of a positron emission tomography (PET) tracer based on CRANAD-102 could as such allow to noninvasively study soluble and protofibrillar species of Aβ in humans. These soluble and protofibrillar species are thought to be responsible to cause AD. Within this work, we successfully 11 C-labeled CRANAD-102 via a Suzuki-Miyaura reaction in a RCС of 48 ± 9%, with a RCP of >96% and a molar activity (A m ) of 25 ± 7 GBq/μmol. Future studies have to be conducted to evaluate if [ 11 C]CRANAD-102 can be used to detect soluble protofibrils in vivo and if [ 11 C]CRANAD-102 can be used to detect AD earlier as possible with current diagnostics., (© 2023 John Wiley & Sons, Ltd.)

Rationale: The psychedelic effects of the traditional Amazonian botanical decoction known as ayahuasca are often attributed to agonism at brain serotonin 5-HT 2A receptors by N,N -dimethyltryptamine (DMT). To reduce first pass metabolism of oral DMT, ayahuasca preparations additionally contain reversible monoamine oxidase A (MAO-A) inhibitors, namely β-carboline alkaloids such as harmine. However, there is lacking biochemical evidence to substantiate this pharmacokinetic potentiation of DMT in brain via systemic MAO-A inhibition. Objectives: We measured the pharmacokinetic profile of harmine and/or DMT in rat brain, and tested for pharmacodynamic effects on brain glucose metabolism and DMT occupancy at brain serotonin 5-HT 2A receptors. Methods: We first measured brain concentrations of harmine and DMT after treatment with harmine and/or DMT at low sub-cutaneous doses (1 mg/kg each) or harmine plus DMT at moderate doses (3 mg/kg each). In the same groups of rats, we also measured ex vivo the effects of these treatments on the availability of serotonin 5-HT 2A receptors in frontal cortex. Finally, we explored effects of DMT and/or harmine (1 mg/kg each) on brain glucose metabolism with [ 18 F]FDG-PET. Results: Results confirmed that co-administration of harmine inhibited the formation of the DMT metabolite indole-3-acetic acid (3-IAA) in brain, while correspondingly increasing the cerebral availability of DMT. However, we were unable to detect any significant occupancy by DMT at 5-HT 2A receptors measured ex vivo , despite brain DMT concentrations as high as 11.3 µM. We did not observe significant effects of low dose DMT and/or harmine on cerebral [ 18 F]FDG-PET uptake. Conclusion: These preliminary results call for further experiments to establish the dose-dependent effects of harmine/DMT on serotonin receptor occupancy and cerebral metabolism., Competing Interests: KE, FG, NJ, CB, SP, VS, MH, BQ, and PC have nothing to declare. DD and MS co-founded Reconnect Labs, an academic spin-off at the University of Zurich. MP has an ongoing research collaboration with Compass Pathways Ltd. unrelated to the present study., (Copyright © 2023 Egger, Gudmundsen, Jessen, Baun, Poetzsch, Shalgunov, Herth, Quednow, Martin-Soelch, Dornbierer, Scheidegger, Cumming and Palner.)

Quality of life is often reduced in patients with sleep-wake disorders. Insomnia is commonly treated with benzodiazepines, despite their well-known side effects. Pellotine ( 1 ), a Lophophora alkaloid, has been reported to have short-acting sleep-inducing properties in humans. In this study, we set out to evaluate various in vitro and in vivo properties of 1 . We demonstrate that 1 undergoes slow metabolism; e.g. in mouse liver microsomes 65% remained, and in human liver microsomes virtually no metabolism was observed after 4 h. In mouse liver microsomes, two phase I metabolites were identified: 7-desmethylpellotine and pellotine- N -oxide. In mice, the two diastereomers of pellotine -O -glucuronide were additionally identified as phase II metabolites. Furthermore, we demonstrated by DESI-MSI that 1 readily enters the central nervous system of rodents. Furthermore, radioligand-displacement assays showed that 1 is selective for the serotonergic system and in particular the serotonin (5-HT) 1D , 5-HT 6 , and 5-HT 7 receptors, where it binds with affinities in the nanomolar range (117, 170, and 394 nM, respectively). Additionally, 1 was functionally characterized at 5-HT 6 and 5-HT 7 , where it was found to be an agonist at the former (EC 50 = 94 nM, E max = 32%) and an inverse agonist at the latter (EC 50 = 291 nM, E max = -98.6). Finally, we demonstrated that 1 dose-dependently decreases locomotion in mice, inhibits REM sleep, and promotes sleep fragmentation. Thus, we suggest that pellotine itself, and not an active metabolite, is responsible for the hypnotic effects and that these effects are possibly mediated through modulation of serotonergic receptors., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

Psilocybin (a classic serotonergic psychedelic drug) has received appraisal for use in psychedelic-assisted therapy of several psychiatric disorders. A less explored topic concerns the use of repeated low doses of psychedelics, at a dose that is well below the psychedelic dose used in psychedelic-assisted therapy and often referred to as microdosing. Psilocybin microdose users frequently report increases in mental health, yet such reports are often highly biased and vulnerable to placebo effects. Here we establish and validate a psilocybin microdose-like regimen in rats with repeated low doses of psilocybin administration at a dose derived from occupancy at rat brain 5-HT 2A receptors in vivo. The rats tolerated the repeated low doses of psilocybin well and did not manifest signs of anhedonia, anxiety, or altered locomotor activity. There were no deficits in pre-pulse inhibition of the startle reflex, nor did the treatment downregulate or desensitize the 5-HT 2A receptors. However, the repeated low doses of psilocybin imparted resilience against the stress of multiple subcutaneous injections, and reduced the frequency of self-grooming, a proxy for human compulsive actions, while also increasing 5-HT 7 receptor expression and synaptic density in the paraventricular nucleus of the thalamus. These results establish a well-validated regimen for further experiments probing the effects of repeated low doses of psilocybin. Results further substantiate anecdotal reports of the benefits of psilocybin microdosing as a therapeutic intervention, while pointing to a possible physiological mechanism., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

A method to detect and quantify aggregated α-synuclein (αSYN) fibrils in vivo would drastically impact the current understanding of multiple neurodegenerative diseases, revolutionizing their diagnosis and treatment. Several efforts have produced promising scaffolds, but a notable challenge has hampered the establishment of a clinically successful αSYN positron emission tomography (PET) tracer: the requirement of high selectivity over the other misfolded proteins amyloid β (Aβ) and tau. By designing and screening a library of 2-styrylbenzothiazoles based on the selective fluorescent probe RB1 , this study aimed at developing a selective αSYN PET tracer. [ 3 H]PiB competition binding assays identified PFSB ( K i = 25.4 ± 2.3 nM) and its less lipophilic analogue MFSB , which exhibited enhanced affinity to αSYN ( K i = 10.3 ± 4.7 nM) and preserved selectivity over Aβ. The two lead compounds were labeled with fluorine-18 and evaluated using in vitro autoradiography on human brain slices, where they demonstrated up to 4-fold increased specific binding in MSA cases compared to the corresponding control, reasonably reflecting selective binding to αSYN pathology. In vivo PET imaging showed [ 18 F] MFSB successfully crosses the blood-brain barrier (BBB) and is taken up in the brain (SUV = 1.79 ± 0.02). Although its pharmacokinetic profile raises the need for additional structural optimization, [ 18 F] MFSB represents a critical step forward in the development of a successful αSYN PET tracer by overcoming the major challenge of αSYN/Aβ selectivity., Competing Interests: The authors declare the following competing financial interest(s): The following EU patent application has been filed: 5402P670EP., (© 2023 The Authors. Published by American Chemical Society.)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) Omicron variant sub-lineages spread rapidly worldwide, mostly due to their immune-evasive properties. This has put a significant part of the population at risk for severe disease and underscores the need for effective anti-SARS-CoV-2 agents against emergent strains in vulnerable patients. Camelid nanobodies are attractive therapeutic candidates due to their high stability, ease of large-scale production, and potential for delivery via inhalation. Here, we characterize the receptor binding domain (RBD)-specific nanobody W25 and show superior neutralization activity toward Omicron sub-lineages in comparison to all other SARS-CoV2 variants. Structure analysis of W25 in complex with the SARS-CoV2 spike glycoprotein shows that W25 engages an RBD epitope not covered by any of the antibodies previously approved for emergency use. In vivo evaluation of W25 prophylactic and therapeutic treatments across multiple SARS-CoV-2 variant infection models, together with W25 biodistribution analysis in mice, demonstrates favorable pre-clinical properties. Together, these data endorse W25 for further clinical development., Competing Interests: D.W., K.C., and P.R.Y are listed as inventors of ‘Molecular Clamp’ patent, US 2020/0040042., (© 2023.)

A technique to image α-synuclein (αSYN) fibrils in vivo is an unmet scientific and clinical need that would represent a transformative tool in the understanding, diagnosis, and treatment of various neurodegenerative diseases. Several classes of compounds have shown promising results as potential PET tracers, but no candidate has yet exhibited the affinity and selectivity required to reach clinical application. We hypothesized that the application of the rational drug design technique of molecular hybridization to two promising lead scaffolds could enhance the binding to αSYN up to the fulfillment of those requirements. By combining the structures of SIL and MODAG tracers, we developed a library of diarylpyrazoles (DAPs). In vitro evaluation through competition assays against [ 3 H]SIL26 and [ 3 H]MODAG-001 showed the novel hybrid scaffold to have preferential binding affinity for amyloid β (Aβ) over αSYN fibrils. A ring-opening modification on the phenothiazine building block to produce analogs with increased three-dimensional flexibility did not result in an improved αSYN binding but a complete loss of competition, as well as a significant reduction in Aβ affinity. The combination of the phenothiazine and the 3,5-diphenylpyrazole scaffolds into DAP hybrids did not generate an enhanced αSYN PET tracer lead compound. Instead, these efforts identified a scaffold for promising Aβ ligands that may be relevant to the treatment and monitoring of Alzheimer's disease (AD).

'Pretargeting' led to increased target-to-background ratios of nanomedicines in short timeframes. However, clearing or masking agents are needed to reach the full potential of pretargeted approaches. This review gives an overview of clearing and masking agents employed in pretargeting strategies in both preclinical and clinical settings and discusses how these agents work.

Aptamers are promising targeting agents for imaging and therapy of numerous diseases, including cancer. However, a significant shortcoming of aptamers is their poor stability and fast excretion, limiting their application in vivo. Common strategies to overcome these challenges is to chemically modify aptamers in order to increase their stability and/or to apply formulation technologies such as conjugating them to polymers or nanocarriers in order to increase their circulation half-life. This is expected to result in improved cellular uptake or retention to passively targeted nanomedicines. Herein, we report a modular conjugation strategy based on click chemistry between functionalized tetrazines and trans -cyclooctene (TCO), for the modification of high molecular weight hyperbranched polyglycerol (HPG) with sgc8 aptamer, fluorescent dyes, and 111 In. Our data indicate strong affinity of sgc8 against a range of solid tumor-derived cell lines that have previously not been tested with this aptamer. Nevertheless, nonspecific uptake of scrambled ssDNA-functionalized HPG in cells highlights inherent challenges of aptamer-targeted probes that remain to be solved for clinical translation. We validate HPG-sgc8 as a nontoxic nanoprobe with high affinity against MDA-MB-468 breast and A431 lung cancer cells and show significantly increased plasma stability compared to free sgc8. In vivo quantitative SPECT/CT imaging indicates EPR-mediated tumor uptake of HPG-sgc8 and nontargeted or scrambled ssDNA-conjugated HPG but no statistically significant difference between these formulations in terms of total tumor uptake or retention. Our study emphasizes the need for stringent controls and quantification in the evaluation of aptamer-targeted probes. For this purpose, our versatile synthesis strategy provides a simple approach for the design and evaluation of long-circulating aptamer-conjugated nanoformulations., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Tucatinib is a selective human epidermal growth factor receptor 2 (HER2) tyrosine kinase inhibitor approved by the U.S. Food and Drug Administration (FDA) in April 2020 for HER2-positive lesions in metastatic breast cancer patients, including CNS metastases. In this article, we attempted to develop the first small molecule, blood-brain-barrier (BBB) penetrant HER2 PET imaging probe based on tucatinib. [ 11 C]tucatinib was synthesized via a Stille-coupling from the respective trimethylstannyl precursor and its biodistribution was evaluated in NMRI nude mice bearing HER2-overexpressing human ovarian cancer cells (SKOV-3). No significant tumor accumulation was observed despite its high affinity for HER-2 receptors (IC 50  = 6.9 nM). High liver and intestinal uptake indicate that [ 11 C]tucatinib is too lipophilic to be used as a tumor targeting PET tracer. Therefore, chemical modifications of [ 11 C]tucatinib are needed to increase the polarity for tumor imaging. Tucatinib as an FDA approved drug is still an interesting platform to develop the first small molecule HER2-selective PET tracer. The study highlights the differences between a drug, which needs to be effective, and an imaging agent, which is dependent on contrast., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Pretargeting imaging has gained a lot of prominence, due to its excellent bioorthogonality and improved imaging contrast compared to conventional imaging. A new iodo tetrazine (Tz) derivative has been synthesized and further developed into the corresponding iodine-125 ( 125 I) analog (12), via the trimethylstannane precursor. Radiolabeling with either N-chlorosuccinimide or chloramine-T, in either MeCN or MeOH proceeded with a radiochemical conversion (RCC) of >80%. Subsequent deprotection only proved successful, among the tested conditions, when the radiolabeled Tz was stirred in 6-M HCl (aq.) at 60°C for 2.5 h. To the best of our knowledge, this is the first H-tetrazine labeled with iodine. In vivo investigations on the pretargeting ability of 12 are currently under way., (© 2022 The Authors. Journal of Labelled Compounds and Radiopharmaceuticals published by John Wiley & Sons Ltd.)

Human serum albumin (HSA) has been shown to be a promising tumor targeting vector and target for generating theranostics by bioconjugation. Unstable chemical conjugation to HSA via a cysteine (Cys34) by reversible Michael additions is most commonly applied for this purpose. Herein, we describe utilization of our recently developed site-selective irreversible S N Ar conjugation to Cys34 using perfluorobenzene sulfonyl derivatives to introduce a trans -cyclooctene (TCO) handle. The TCO could then be bioorthogonally ligated within minutes through an inverse-electron demand Diels-Alder reaction (IEDDA) to tetrazines (Tzs) containing a radionuclide. The methodology opens up a wide range of chemistries including pretargeting, 'click-to-release' tumor selective drug delivery or ultra-fast and complete conjugation of any drug. The proof-of-principle study demonstrated that the conjugation chemistry is feasible, robust and easy to carry out, being promising for pretargeted imaging and therapy studies as well as selective drug delivery using HSA., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Tetrazine (Tz)-trans-cyclooctene (TCO) ligation is an ultra-fast and highly selective reaction and it is particularly suited to label biomolecules under physiological conditions. As such, a 3 H-Tz based synthon would have wide applications for in vitro/ex vivo assays. In this study, we developed a 3 H-labeled Tz and characterized its potential for application to pretargeted autoradiography. Several strategies were explored to synthesize such a Tz. However, classical approaches such as reductive halogenation failed. For this reason, we designed a Tz containing an aldehyde and explored the possibility of reducing this group with NaBT 4 . This approach was successful and resulted in [ 3 H]-(4-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)phenyl)methan-t-ol with a radiochemical yield of 22 %, a radiochemical purity of 96 % and a molar activity of 0.437 GBq/μmol (11.8 Ci/mmol). The compound was successfully applied to pretargeted autoradiography. Thus, we report the synthesis of the first 3 H-labeled Tz and its successful application as a labeling building block., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Pretargeting is a powerful nuclear imaging strategy to achieve enhanced imaging contrast for nanomedicines and reduce the radiation burden to healthy tissue. Pretargeting is based on bioorthogonal chemistry. The most attractive reaction for this purpose is currently the tetrazine ligation, which occurs between trans -cyclooctene (TCO) tags and tetrazines (Tzs). Pretargeted imaging beyond the blood-brain barrier (BBB) is challenging and has not been reported thus far. In this study, we developed Tz imaging agents that are capable of ligating in vivo to targets beyond the BBB. We chose to develop 18 F-labeled Tzs as they can be applied to positron emission tomography (PET) - the most powerful molecular imaging technology. Fluorine-18 is an ideal radionuclide for PET due to its almost ideal decay properties. As a non-metal radionuclide, fluorine-18 also allows for development of Tzs with physicochemical properties enabling passive brain diffusion. To develop these imaging agents, we applied a rational drug design approach. This approach was based on estimated and experimentally determined parameters such as the BBB score, pretargeted autoradiography contrast, in vivo brain influx and washout as well as on peripheral metabolism profiles. From 18 initially developed structures, five Tzs were selected to be tested for their in vivo click performance. Whereas all selected structures clicked in vivo to TCO-polymer deposited into the brain, [ 18 F]18 displayed the most favorable characteristics with respect to brain pretargeting. [ 18 F]18 is our lead compound for future pretargeted neuroimaging studies based on BBB-penetrant monoclonal antibodies. Pretargeting beyond the BBB will allow us to image targets in the brain that are currently not imageable, such as soluble oligomers of neurodegeneration biomarker proteins. Imaging of such currently non-imageable targets will allow early diagnosis and personalized treatment monitoring. This in turn will accelerate drug development and greatly benefit patient care., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

COVID-19, caused by the SARS-CoV-2 virus, has become a global pandemic that is still present after more than two years. COVID-19 is mainly known as a respiratory disease that can cause long-term consequences referred to as long COVID. Molecular imaging of SARS-CoV-2 in COVID-19 patients would be a powerful tool for studying the pathological mechanisms and viral load in different organs, providing insights into the disease and the origin of long-term consequences and assessing the effectiveness of potential COVID-19 treatments. Current diagnostic methods used in the clinic do not allow direct imaging of SARS-CoV-2. In this work, a nanobody (NB) - a small, engineered protein derived from alpacas - and an Fc-fused NB which selectively target the SARS-CoV-2 Spike protein were developed as imaging agents for positron emission tomography (PET). We used the tetrazine ligation to 18 F-label the NB under mild conditions once the NBs were successfully modified with trans- cyclooctenes (TCOs). We confirmed binding to the Spike protein by SDS-PAGE. Dynamic PET scans in rats showed excretion through the liver for both constructs. Future work will evaluate in vivo binding to the Spike protein with our radioligands., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2022 Lopes van den Broek, García-Vázquez, Andersen, Valenzuela-Nieto, Shalgunov, Battisti, Schwefel, Modhiran, Kramer, Cheuquemilla, Jara, Salinas-Varas, Amarilla, Watterson, Rojas-Fernandez and Herth.)

Combining nanotechnology and bioorthogonal chemistry for theranostic strategies offers the possibility to develop next generation nanomedicines. These materials are thought to increase therapeutic outcome and improve current cancer management. Due to their size, nanomedicines target tumors passively. Thus, they can be used for drug delivery purposes. Bioorthogonal chemistry allows for a pretargeting approach. Higher target-to-background drug accumulation ratios can be achieved. Pretargeting can also be used to induce internalization processes or trigger controlled drug release. Colloidal gold nanoparticles (AuNPs) have attracted widespread interest as drug delivery vectors within the last decades. Here, we demonstrate for the first time the possibility to successfully ligate AuNPs in vivo to pretargeted monoclonal antibodies. We believe that this possibility will facilitate the development of AuNPs for clinical use and ultimately, improve state-of-the-art patient care., (© 2022 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Background: A positron emission tomography (PET) radiotracer to neuroimage α-synuclein aggregates would be a crucial addition for early diagnosis and treatment development in disorders such as Parkinson's disease, where elevated aggregate levels are a histopathological hallmark. The radiotracer (d 3 )-[ 11 C]MODAG-001 has recently shown promise for visualization of α-synuclein pre-formed fibrils (α-PFF) in rodents. We here test the radiotracer in a pig model where proteins are intracerebrally injected immediately before scanning. Four pigs were injected in one hemisphere with 150 μg α-PFF, and in the other hemisphere, either 75 μg α-PFF or human brain homogenate from either dementia with Lewy bodies (DLB) or Alzheimer's disease (AD) was injected. All pigs underwent one or two (d 3 )-[ 11 C]MODAG-001 PET scans, quantified with the non-invasive Logan graphical analysis using the occipital cortex as a reference region., Results: The α-PFF and AD homogenate injected brain regions had high uptake of (d 3 )-[ 11 C]MODAG-001 compared to the occipital cortex or cerebellum. BP ND values in 150 μg α-PFF injected regions was 0.78, and in the AD homogenate injected regions was 0.73. By contrast, the DLB homogenate injected region did not differ in uptake and clearance compared to the reference regions. The time-activity curves and BP ND values in the 150 μg and 75 μg injected regions of α-PFFs show a dose-dependent effect, and the PET signal could be blocked by pretreatment with unlabeled MODAG-001., Conclusion: We find that both α-PFF and AD brain homogenates give rise to increased binding of (d 3 )-[ 11 C]MODAG-001 when injected into the pig brain. Despite its limited specificity for cerebral α-synuclein pathology, (d 3 )-[ 11 C]MODAG-001 shows promise as a lead tracer for future radiotracer development., Competing Interests: Declaration of competing interest Lundbeck A/S, Denmark provided the α-synuclein preformed fibrils as part of the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 813528. However, they had no other financial interest in the project. GMK received honoraria as a speaker and consultant for Sage Pharmaceuticals/Biogen and Sanos A/S. All other authors declare no conflict of interest., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The blood-brain barrier (BBB) is a protective and semipermeable border of endothelial cells that prevents toxins and foreign bodies to enter and damage the brain. Unfortunately, the BBB also hampers the development of pharmaceuticals targeting receptors, enzymes, or other proteins that lie beyond this barrier. Especially large molecules, such as monoclonal antibodies (mAbs) or nanoparticles, are prevented to enter the brain. The limited passage of these molecules partly explains why nanomedicines - targeting brain diseases - have not made it into the clinic to a great extent. As nanomedicines can target a wide range of targets including protein isoforms and oligomers or potentially deliver cytotoxic drugs safely to their targets, a pathway to smuggle nanomedicines into the brain would allow to treat brain diseases that are currently considered 'undruggable'. In this review, strategies to transport nanomedicines over the BBB will be discussed. Their challenges and opportunities will be highlighted with respect to their use for molecular imaging or therapies. Several strategies have been explored for this thus far. For example, carrier-mediated and receptor-mediated transcytosis (RMT), techniques to disrupt the BBB, nasal drug delivery or administering nanomedicines directly into the brain have been explored. RMT has been the most widely and successfully explored strategy. Recent work on the use of focused ultrasound based BBB opening has shown great promise. For example, successful delivery of mAbs into the brain has been achieved, even in a clinical setting. As nanomedicines bear the potential to treat incurable brain diseases, drug delivery technologies that can deliver nanomedicines into the brain will play an essential role for future treatment options., Competing Interests: Declaration of competing interest There is no conflict of interests., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Pretargeting is a promising nuclear imaging technique that allows for the usage of antibodies (Abs) with enhanced imaging contrast and reduced patient radiation burden. It is based on bioorthogonal chemistry with the tetrazine ligation-a reaction between trans -cyclooctenes (TCOs) and tetrazines (Tzs)-currently being the most popular reaction due to its high selectivity and reactivity. As Abs can be designed to bind specifically to currently 'undruggable' targets such as protein isoforms or oligomers, which play a crucial role in neurodegenerative diseases, pretargeted imaging beyond the BBB is highly sought after, but has not been achieved yet. A challenge in this respect is that large molecules such as Abs show poor brain uptake. Uptake can be increased by receptor mediated transcytosis; however, it is largely unknown if the achieved brain concentrations are sufficient for pretargeted imaging. In this study, we investigated whether the required concentrations are feasible to reach. As a model Ab, we used the bispecific anti-amyloid beta (Aβ) anti-transferrin receptor (TfR) Ab 3D6scFv8D3 and conjugated it to a different amount of TCOs per Ab and tested different concentrations in vitro. With this model in hand, we estimated the minimum required TCO concentration to achieve a suitable contrast between the high and low binding regions. The estimation was carried out using pretargeted autoradiography on brain sections of an Alzheimer's disease mouse model. Biodistribution studies in wild-type (WT) mice were used to correlate how different TCO/Ab ratios alter the brain uptake. Pretargeted autoradiography showed that increasing the number of TCOs as well as increasing the TCO-Ab concentration increased the imaging contrast. A minimum brain concentration of TCOs for pretargeting purposes was determined to be 10.7 pmol/g in vitro. Biodistribution studies in WT mice showed a brain uptake of 1.1% ID/g using TCO-3D6scFv8D3 with 6.8 TCO/Ab. According to our estimations using the optimal parameters, pretargeted imaging beyond the BBB is not a utopia. Necessary brain TCO concentrations can be reached and are in the same order of magnitude as required to achieve sufficient contrast. This work gives a first estimate that pretargeted imaging is indeed possible with antibodies. This could allow the imaging of currently 'undruggable' targets and therefore be crucial to monitor (e.g., therapies for intractable neurodegenerative diseases).

Alzheimer's disease (AD) is the most common neurodegenerative disease, with an increasing prevalence. Currently, there is no ideal diagnostic molecular imaging agent for diagnosing AD. Antibodies (Abs) have been proposed to close this gap as they can bind selectively and with high affinity to amyloid β (Aβ)-one of the molecular hallmarks of AD. Abs can even be designed to selectively bind Aβ oligomers or isoforms, which are difficult to target with small imaging agents. Conventionally, Abs must be labeled with long-lived radionuclides which typically results in in high radiation burden to healthy tissue. Pretargeted imaging could solve this challenge as it allows for the use of short-lived radionuclides. To develop pretargeted imaging tools that can enter the brain, AD mouse models are useful as they allow testing of the imaging approach in a relevant animal model that could predict its clinical applicability. Several mouse models for AD have been developed with different characteristics. Commonly used models are: 5xFAD, APP/PS1 and tg-ArcSwe transgenic mice. In this study, we aimed to identify which of these models were best suited to investigate pretargeted imaging approaches beyond the blood brain barrier. We evaluated this by pretargeted autoradiography using the Aβ-targeting antibody 3D6 and an 111 In-labeled Tz. Evaluation criteria were target-to-background ratios and accessibility. APP/PS1 mice showed Aβ accumulation in high and low binding brain regions and is as such less suitable for pretargeted purposes. 5xFAD and tg-ArcSwe mice showed similar uptake in high binding regions whereas low uptake in low binding regions and are better suited to evaluate pretargeted imaging approaches. 5xFAD mice are advantaged over tg-ArcSwe mice as pathology can be traced early (6 months compared to 18 months of age) and as 5xFAD mice are commercially available., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2022 Lopes van den Broek, Sehlin, Andersen, Aldana, Beschörner, Nedergaard, Knudsen, Syvänen and Herth.)

Background: The development of radiopharmaceuticals requires extensive evaluation before they can be applied in a diagnostic or therapeutic setting in Nuclear Medicine. Chemical, radiochemical, and pharmaceutical parameters must be established and verified to ensure the quality of these novel products., Main Body: To provide supportive evidence for the expected human in vivo behaviour, particularly related to safety and efficacy, additional tests, often referred to as "non-clinical" or "preclinical" are mandatory. This document is an outcome of a Technical Meeting of the International Atomic Energy Agency. It summarises the considerations necessary for non-clinical studies to accommodate the regulatory requirements for clinical translation of radiopharmaceuticals. These considerations include non-clinical pharmacology, radiation exposure and effects, toxicological studies, pharmacokinetic modelling, and imaging studies. Additionally, standardisation of different specific clinical applications is discussed., Conclusion: This document is intended as a guide for radiopharmaceutical scientists, Nuclear Medicine specialists, and regulatory professionals to bring innovative diagnostic and therapeutic radiopharmaceuticals into the clinical evaluation process in a safe and effective way., (© 2022. The Author(s).)

Astatine-211 ( 211 At) is one of the most promising α-emitters for targeted alpha therapy, especially of cancer metastases. However, the lack of a stable isotope, frequent in vivo deastatination, and limited radiochemical knowledge makes it challenging to apply. Here, we report a new strategy for radiolabeling the lipophilic core of polymeric micelles (PMs) with covalently bound 211 At. The PMs were radiolabeled via either an indirect synthon-based method or directly on the amphipathic block copolymer. The radiochemistry was optimized with iodine-125 ( 125 I) and then adapted for 211 At, enabling the use of both elements as a potential theranostic pair. PMs that were core-radiolabeled with both 125 I or 211 At were prepared and characterized, based on a PEG(5k)-PLGA(10k) co-polymer. The stability of the radiolabeled PMs was evaluated in mouse serum for 21 h, showing radiochemical stability above 85%. After in vivo evaluation of the 211 At- labeled PMs, 4-5 % ID/g of the 211 At could still be detected in the blood, showing a promising in vivo stability of the PMs. Further, 211 At-labeled PMs accumulated in the spleen (20-30 %ID/g) and the liver (2.5- 5.5 %ID/g), along with some detection of 211 At in the thyroid (3.5-9 %ID/g). This led to the hypothesis that deastatination takes place in the liver, whereas good stability of the 211 At core-radiolabel was observed in the blood., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Radiolabeling of tetrazines has gained increasing attention due to their important role in pretargeted imaging or therapy. The most commonly used radionuclide in PET imaging is fluorine-18. For this reason, we have recently developed a method which enables the direct aromatic 18 F-fluorination of tetrazines using stannane precursors through copper-mediated fluorinations. Herein, we further optimized this labeling procedure. 3-(3-fluorophenyl)-1,2,4,5-tetrazine was chosen for this purpose because of its high reactivity and respective limited stability during the labeling process. By optimizing parameters such as elution conditions, precursor amount, catalyst, time or temperature, the radiochemical yield (RCY) could be increased by approximately 30%. These conditions were then applied to optimize the RCY of a recently successfully developed and promising pretargeting imaging agent. This agent could be isolated in a decay corrected RCY of 14 ± 3% and Am of 201 ± 30 GBq/µmol in a synthesis time of 70 min. Consequently, the RCY increased by 27%.

Functionalization of macromolecules (antibodies, polymers, nanoparticles) with click-reactive groups greatly enhances the versatility of their potential applications. Click chemistry based on tetrazine - trans-cyclooctene (TCO) ligation is especially promising and is already widely applied for pretargeted imaging and therapy. Indirect radiolabeling of TCO-functionalized macromolecules with substoichiometric amounts of radioactive tetrazines is a convenient way to monitor the fate of those macromolecules by means of positron emission tomography (PET) imaging after their administration into the test subject. In this work, the preparation is reported of TCO-containing graft copolymers, namely PeptoBrushes (polyglutamic acid-graft-polysarcosine), novel [ 11 C]carboxylated tetrazines, and their combined use in radiolabeling the polymer by inverse electron demand Diels Alder reaction, to investigate it is potential for an application in pretarget imaging or injectable brachytherapy. The procedure for [ 11 C]tetrazine production is easy and scalable, while indirect TCO-PeptoBrushes labeling with these [ 11 C]tetrazines is mild, fast, and quantitative. This strategy allows facile 11 C-labeling of diverse TCO-functionalized macromolecules, so that their localization and distribution shortly after injection can be assessed by PET., (© 2021 The Authors. Macromolecular Rapid Communications published by Wiley-VCH GmbH.)

Tetrazine ligation has gained interest as a bio-orthogonal chemistry tool within the last decade. In nuclear medicine, tetrazine ligation is currently being explored for pretargeted approaches, which have the potential to revolutionize state-of-the-art theranostic strategies. Pretargeting has been shown to increase target-to-background ratios for radiopharmaceuticals based on nanomedicines, especially within early timeframes. This allows the use of radionuclides with short half-lives which are more suited for clinical applications. Pretargeting bears the potential to increase the therapeutic dose delivered to the target as well as reduce the respective dose to healthy tissue. Combined with the possibility to be applied for diagnostic imaging, pretargeting could be optimal for theranostic approaches. In this review, we highlight efforts that have been made to radiolabel tetrazines with an emphasis on imaging.

Tetrazines (Tz) have been applied as bioorthogonal agents for various biomedical applications, including pretargeted imaging approaches. In radioimmunoimaging, pretargeting increases the target-to-background ratio while simultaneously reducing the radiation burden. We have recently reported a strategy to directly 18 F-label highly reactive tetrazines based on a 3-(3-fluorophenyl)-Tz core structure. Herein, we report a kinetic study on this versatile scaffold. A library of 40 different tetrazines was prepared, fully characterized, and investigated with an emphasis on second-order rate constants for the reaction with trans -cyclooctene (TCO). Our results reveal the effects of various substitution patterns and moreover demonstrate the importance of measuring reactivities in the solvent of interest, as click rates in different solvents do not necessarily correlate well. In particular, we report that tetrazines modified in the 2-position of the phenyl substituent show high intrinsic reactivity toward TCO, which is diminished in aqueous systems by unfavorable solvent effects. The obtained results enable the prediction of the bioorthogonal reactivity and thereby facilitate the development of the next generation of substituted aryltetrazines for in vivo applications.

Pretargeted PET imaging is an emerging and fast-developing method to monitor immuno-oncology strategies. Currently, tetrazine ligation is considered the most promising bioorthogonal reaction for pretargeting in vivo. Recently, we have developed a method to 18 F-label ultrareactive tetrazines by copper-mediated fluorinations. However, bispyridyl tetrazines-one of the most promising structures for in vivo pretargeted applications-were inaccessible using this strategy. We believed that our successful efforts to 18 F-label H-tetrazines using low basic labeling conditions could also be used to label bispyridyl tetrazines via aliphatic nucleophilic substitution. Here, we report the first direct 18 F-labeling of bispyridyl tetrazines, their optimization for in vivo use, as well as their successful application in pretargeted PET imaging. This strategy resulted in the design of [ 18 F] 45 , which could be labeled in a satisfactorily radiochemical yield (RCY = 16%), molar activity (A m = 57 GBq/µmol), and high radiochemical purity (RCP > 98%). The [ 18 F] 45 displayed a target-to-background ratio comparable to previously successfully applied tracers for pretargeted imaging. This study showed that bispyridyl tetrazines can be developed into pretargeted imaging agents. These structures allow an easy chemical modification of 18 F-labeled tetrazines, paving the road toward highly functionalized pretargeting tools. Moreover, bispyridyl tetrazines led to near-instant drug release of iTCO-tetrazine-based 'click-to-release' reactions. Consequently, 18 F-labeled bispyridyl tetrazines bear the possibility to quantify such release in vivo in the future.

Introduction: Radiotherapy of cancer requires both alpha- and beta-particle emitting radionuclides, as these radionuclide types are efficient at destroying different types of tumors. Both classes of radionuclides require a vehicle, such as an antibody or a polymer, to be delivered and retained within the tumor. Polyglutamic acid (pGlu) is a polymer that has proven itself effective as a basis of drug-polymer conjugates in the clinic, while its derivatives have been used for pretargeted tumor imaging in a research setup. trans-Cyclooctene (TCO) modified pGlu is suitable for pretargeted imaging or therapy, as well as for intratumoral radionuclide therapy. In all cases, it becomes indirectly radiolabeled via the bioorthogonal click reaction with the tetrazine (Tz) molecule carrying the radionuclide. In this study, we report the radiolabeling of TCO-modified pGlu with either lutetium-177 ( 177 Lu), a beta-particle emitter, or actinium-225 ( 225 Ac), an alpha-particle emitter, using the click reaction between TCO and Tz., Methods: A panel of Tz derivatives containing a metal ion binding chelator (DOTA or macropa) connected to the Tz moiety directly or through a polyethylene glycol (PEG) linker was synthesized and tested for their ability to chelate 177 Lu and 225 Ac, and click to pGlu-TCO. Radiolabeled 177 Lu-pGlu and 225 Ac-pGlu were isolated by size exclusion chromatography. The retention of 177 Lu or 225 Ac by the obtained conjugates was investigated in vitro in human serum., Results: All DOTA-modified Tzs efficiently chelated 177 Lu resulting in average radiochemical conversions (RCC) of >75%. Isolated radiochemical yields (RCY) for 177 Lu-pGlu prepared from 177 Lu-Tzs ranged from 31% to 55%. TLC analyses detected <5% unchelated 177 Lu for all 177 Lu-pGlu preparations over six days in human serum. For 225 Ac chelation, optimized RCCs ranged from 61 ± 34% to quantitative for DOTA-Tzs and were quantitative for the macropa-modified Tz (>98%). Isolated radiochemical yields (RCY) for 225 Ac-pGlu prepared from 225 Ac-Tzs ranged from 28% to 51%. For 3 out of 5 225 Ac-pGlu conjugates prepared from DOTA-Tzs, the amount of unchelated 225 Ac stayed below 10% over six days in human serum, while 225 Ac-pGlu prepared from macropa-Tz showed a steady release of up to 37% 225 Ac., Conclusion: We labeled TCO-modified pGlu polymers with alpha- and beta-emitting radionuclides in acceptable RCYs. All 177 Lu-pGlu preparations and some 225 Ac-pGlu preparations showed excellent stability in human plasma. Our work shows the potential of pGlu as a vehicle for alpha- and beta-radiotherapy of tumors and demonstrated the usefulness of Tz ligation for indirect radiolabeling., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Fluorine-18 displays almost ideal decay properties for positron emission tomography (PET) and allows for large scale production. As such, simplified methods to radiolabel peptides with fluorine-18 are highly warranted. Chelation of aluminium fluoride-18 toward specific peptides represents one method to achieve this. With the current methods, chelation of aluminium fluoride-18 can be achieved using NOTA-conjugated peptides. However, the heating to 90-100◦C that is required for this chelation approach may be deleterious to the targeting moiety of the probe. Recently, a new chelator, RESCA1, was developed allowing Al 18 F chelation at room temperature. Here, we optimize the labeling procedure enabling high chelation efficacy of fluoride-18 at 22◦C, even at full batch labeling. The optimized procedure was tested by Al 18 F-labeling of RESCA1-AE105-a uPAR targeting peptide. NOTA-AE105 was also labeled with Al 18 F, and the two peptides were compared head-to-head. [ 18 F]AlF-NOTA-AE105 and [ 18 F]AlF-RESCA1-AE105 could be produced in equal radiochemical yields (RCY), radiochemical purities (RCP) and molar activities. Additionally, the two peptides showed comparable binding affinity to uPAR and uptake in cells expressing the uPAR, when evaluated in vitro . Overall, we found that the performances of [ 18 F]AlF-NOTA-AE105 and [ 18 F]AlF-RESCA1-AE105 were grossly comparable, but importantly RESCA1 can be labeled with aluminium fluoride-18 at 22◦C. Consequently, this study showed that RESCA1 is superior to NOTA with respect to Al 18 F chelation of temperature sensitive molecules, such as thermolabile peptides and proteins as well as that full batch chelation of RESCA1 with fluoride-18 is possible., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jeppesen, Simón, Torp, Knudsen, Leth, Crestey, Ploug, Jørgensen, Madsen, Herth and Kjaer.)

Parkinson's disease (PD) is caused by progressive neurodegeneration and characterised by motor dysfunction. Neurodegeneration of dopaminergic neurons also causes aberrations within the cortico-striato-thalamo-cortical (CSTC) circuit, which has been hypothesised to lead to non-motor symptoms such as depression. Individuals with PD have both lower synaptic density and changes in neuronal metabolic function in the basal ganglia, as measured using [ 11 C]UCB-J and [ 18 F]FDG positron emission tomography (PET), respectively. However, the two radioligands have not been directly compared in the same PD subject or in neurodegeneration animal models. Here, we investigate [ 11 C]UCB-J binding and [ 18 F]FDG uptake in the CSTC circuit following a unilateral dopaminergic lesion in rats and compare it to sham lesioned rats. Rats received either a unilateral injection of 6-hydroxydopamine (6-OHDA) or saline in the medial forebrain bundle and rostral substantia nigra ( n = 4/group). After 3 weeks, all rats underwent two PET scans using [ 18 F]FDG, followed by [ 11 C]UCB-J on a separate day. [ 18 F]FDG uptake and [ 11 C]UCB-J binding were both lower in the ipsilateral striatal regions compared to the contralateral regions. Using [ 11 C]UCB-J, we could detect an 8.7% decrease in the ipsilateral ventral midbrain, compared to a 2.9% decrease in ventral midbrain using [ 18 F]FDG. Differential changes between hemispheres for [ 11 C]UCB-J and [ 18 F]FDG outcomes were also evident in the CSTC circuit's cortical regions, especially in the orbitofrontal cortex and medial prefrontal cortex where higher synaptic density yet lower neuronal metabolic function was observed, following lesioning. In conclusion, [ 11 C]UCB-J and [ 18 F]FDG PET can detect divergent changes following a dopaminergic lesion in rats, especially in cortical regions that are not directly affected by the neurotoxin. These results suggest that combined [ 11 C]UCB-J and [ 18 F]FDG scans could yield a better picture of the heterogeneous cerebral changes in neurodegenerative disorders., Competing Interests: MP: Compass Pathways Plc (research collaboration), GK: H. Lundbeck A/S (research collaboration), Compass Pathways Plc (research collaboration), Elysis (research collaboration), Novo Nordisk, Novozymes, Chr. Hansen (stockholder), Sage Therapeutics and Sanos (Advisor). GK is currently the president of the European College of Neuropsychopharmacology. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Raval, Gudmundsen, Juhl, Andersen, Speth, Videbæk, Petersen, Mikkelsen, Fisher, Herth, Plavén-Sigray, Knudsen and Palner.)

Parkinson's disease (PD) is a neurodegenerative disorder that affects millions of people worldwide. Two hallmarks of PD are the accumulation of alpha-synuclein and the loss of dopaminergic neurons in the brain. There is no cure for PD, and all existing treatments focus on alleviating the symptoms. PD diagnosis is also based on the symptoms, such as abnormalities of movement, mood, and cognition observed in the patients. Molecular imaging methods such as magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), and positron emission tomography (PET) can detect objective alterations in the neurochemical machinery of the brain and help diagnose and study neurodegenerative diseases. This review addresses the application of functional MRI, PET, and SPECT in PD patients. We provide an overview of the imaging targets, discuss the rationale behind target selection, the agents (tracers) with which the imaging can be performed, and the main findings regarding each target's state in PD. Molecular imaging has proven itself effective in supporting clinical diagnosis of PD and has helped reveal that PD is a heterogeneous disorder, which has important implications for the development of future therapies. However, the application of molecular imaging for early diagnosis of PD or for differentiation between PD and atypical parkinsonisms has remained challenging. The final section of the review is dedicated to new imaging targets with which one can detect the PD-related pathological changes upstream from dopaminergic degeneration. The foremost of those targets is alpha-synuclein. We discuss the progress of tracer development achieved so far and challenges on the path toward alpha-synuclein imaging in humans., (© 2021 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Pretargeted imaging of nanomedicines have attracted considerable interest because it has the potential to increase imaging contrast while reducing radiation burden to healthy tissue. Currently, the tetrazine ligation is the fastest bioorthogonal reaction for this strategy and, consequently, the state-of-art choice for in vivo chemistry. We have recently identified key properties for tetrazines in pretargeting. We have also developed a method to 18 F-label reactive tetrazines using an aliphatic nucleophilic substitution strategy. Here, we combined this knowledge and developed an 18 F-labeled tetrazine for pretargeted imaging. In order to develop this ligand, a small SAR study was performed. The most promising compound was selected for labeling and subsequent positron-emission-tomography in vivo imaging. Radiolabeling was achieved in satisfactory yields, molar activities, and high radiochemical purities. [ 18 F] 15 displayed favorable pharmacokinetics and remarkable target-to-background ratios-as early as 1 h post injection. We believe that this agent could be a promising candidate for translation into clinical studies.

Purpose: The study demonstrates the use of Desorption Electrospray Ionization mass spectrometry imaging (DESI-MSI) for imaging of the PET tracer compound Cimbi-36 in brain tissue and compares imaging by DESI-MSI to imaging by autoradiography and PET., Procedures: Rats were dosed intraperitoneally with 3 mg/kg of Cimbi-36 and euthanized at t = 5, 10, 15, 30, 60 and 120 min post-injection. The brains were removed, frozen and sectioned, and sagittal sections were imaged by DESI-MSI in positive ion mode. Additionally, brain sections from a non-dosed animal were incubated with 14 C-labelled Cimbi-36 and imaged by autoradiography. Finally, PET images were acquired from an animal dosed with 11 C-labelled Cimbi-36., Results: DESI-MSI and autoradiography images of a sagittal brain sections showed similar distributions of Cimbi-36, with increased abundance in the frontal cortex and choroid plexus, regions which are high in 5-HT 2A and 5-HT 2C receptors. The PET image also showed increased abundance in cortex, but the spatial resolution was clearly inferior to DESI-MSI and autoradiography. The DESI-MSI results showed increased abundance of Cimbi-36 in brain tissue until 15 min, after which the abundance was declining. The PET-tracer was still clearly detectable at t = 120 min. Similar imaging of the kidneys showed the abundance of Cimbi-36 peaking at 30 min. Cimbi-36 was quantified in a t = 15 min brain section by quantitative DESI-MSI, resulting in tissue concentrations of 19.8 μg/g in cortex, 15.4 μg/g in cerebellum and 12.5 μg/g in whole brain., Conclusions: DESI imaging from an in vivo dosing experiment showed distribution of the PET tracer remarkably similar to what was obtained by autoradiography of an in vitro incubation experiment, indicating that the obtained results represent actual binding to certain receptors in the brain. DESI-MSI is suggested as a cost-effective screening tool, which does not rely on labelling of compounds., (© 2021. World Molecular Imaging Society.)